Quantification of tartrate resistant acid phosphatase distribution in mouse tibiae using image analysis

Tartrate resistant acid phosphatase (TRAP) activity of bone is a suitable biochemical marker for osteoclastic bone resorption. Qualitatively, the histochemical distribution of TRAP has been used to identify osteoclasts responsible for bone resorption; however, there have been few attempts to quantify TRAP localization. We describe a method for evaluating bone resorption by quantifying area percentages of positive TRAP localization using image analysis. Mouse tibiae were paraffin embedded following demineralization in disodium ethylenediamine tetraacetic acid. Longitudinal sections of tibia were cut from 15 levels in the left and the right limbs of six mice (180 sections total) and stained for TRAP distribution. Positive TRAP localization was quantified by pixel area count and reported as a percentage of the total tissue area specified. The 1.85 mm² region of interest was placed at the midpoint of the epiphyseal growth plate containing the provisional calcification layer and the primary spongiosa, while excluding cortical bone of each mouse tibia. The percentage of TRAP localization ranged from 0.95 to 1.31% and was not significantly different from level to level or limb to limb in each mouse (p>0.100). Within the same region of interest, an osteoclast count along the bone perimeter also was performed. We demonstrated a strong correlation (r²=0.903) between the conventional histomorphometric osteoclast index and positive TRAP localization, validating the latter as an alternative method to assess bone resorption. Quantitative analysis of TRAP is significant because it allows statistical comparisons between treatment groups, promotes precise pathological diagnoses and facilitates a reference data base that may aid the study of bone related diseases involving increased bone resorption.     

In vivo demonstration of acid phosphatase activity in the rhizosphere of soil-grown plants

For in vivo demonstration of acid phosphatase activity in the rhizosphere of soil-grown plants filter papers were treated with a mixture of 1-naphthyl phosphate as substrate and the diazonium salt Fast Red TR as an indicator. After enzymatic hydrolysis, 1-naphthol forms a red complex with Fast Red TR. This method was applied to 8-day old maize plants and 3-year old Norway spruce plants growing in rhizoboxes in soil under non-sterile conditions. The treated filter paper is placed at the surface of roots and soil and acid phosphatase activity is visualized as a red-coloured root print on the filter paper. The method can be used as a qualitative analysis of acid phosphatase in the rhizosphere. It also allows a rough estimate of phosphatase activity in different root zones.     

Non-destructive methods for demonstrating chemical changes in the rhizosphere II. Application of methods

Chemical changes in the rhizosphere of soil-grown plants are demonstrated by non-destructive techniques based on colour reactions. The following examples are given: FeIII reduction in the rhizosphere of a Hakea species, MnIV reduction in the rhizosphere of chikpea, complexation of Al in the rhizosphere of Norway spruce, and the activity of acid phosphatase in the rhizosphere of maize.     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

Effect of nitrogen forms on growth and chemical changes in the rhizosphere of strawberry plants

The experiment was set up to examine the influence of different nitrogen forms: (NH₄)₂SO₄, Ca(NO₃)₂ or NH₄NO₃ on growth response, root induced pH changes in the rhizosphere, root-borne acid phosphatase activity in strawberry plants cv. Senga Sengana. The plants grown on sandy mineral soil were fertilized with 3 forms of nitrogen, in concentrations of 46 mg N·kg⁻¹ soil. The plants were grown in rhizoboxes with removable plexiglass lids. To ensure the root growth along the plexiglass lids, the rhizoboxes were placed at an angle of about 50° with the lid on the lower side. In case of ammonium supply, the nitrification inhibitor DIDIN was added (10 mg·kg⁻¹ of moist soil) to prevent conversion of ammonium into nitrate. The growth response (roots and shoots) of strawberry plants were determined after 11 weeks of treatment with different N forms. The best development of the root system and shoots (root and shoot dry weight and root length) was obtained, when ammonium nitrate was supplied. It is suggested therefore, that NH₄NO₃ stimulates vegetative growth of strawberry plants cv. Senga Sengana. However, there were no statistical differences in a leaf and flower number of the plants grown under different forms of N-fertilization. Determination of rhizosphere pH, and acid phosphatase activity were executed using non-destructive techniques, which enabled weekly measurement of chemical changes in the rhizosphere. The results revealed that the form of nitrogen supplied had a predominant effect on chemical changes in the rhizosphere of strawberry plants. The highest pH values (average pH 6.8) were measured in the rhizosphere of individual plants supplied with Ca(NO₃)₂. Whereas the lowest pH values (average pH 5.8) were detected in the presence of (NH₄)₂SO₄. The curve of rhizosphere pH measured along individual roots of the plants treated with Ca(NO₃)₂ represents the highest pH values whereas the curve of rhizosphere pH under (NH₄)₂SO₄ treatment had the lowest pH values. The highest activity of acid phosphatase were observed in the rhizosphere of strawberry plants grown in the presence of (NH₄)₂SO₄, at pH 5.8.     

Accurate root length measurement by image analysis

Algorithms for estimating root length by image analysis should lead to results that have no systematic error (bias), be insensitive to preferential root orientation, valid across a wide range of sample sizes and adjust for overlap between roots in samples, to reduce the effort needed in spreading out root systems. We propose a new algorithm that forms a compromise between small bias and robustness (insensitivity to variation in sample size and preferential root orientation), and provide a simple way of dealing with root overlap. Image analysis was performed on a Macintosh computer using the public domain NIH Image program. The digital image of the root was processed to get the thinned image (skeleton). The numbers of orthogonally and diagonally connected pairs of pixels (N _o and N _d, respectively) in the skeleton were counted separately and used for length (L) calculation. A new length calculation equation was introduced so that the effect of orientation on length calculation was minimized; L=[N d ²+(N _d+N _o/2)²]^1/2+N _o/2. The maximum error due to orientation of a single line was evaluated for an ideal line, and the analysis revealed that the new equation was less affected by orientation than previous equations. Copper wire and rice (Oryza sativa L.) roots containing both primary and fine secondary root were measured manually and with image analysis. The two methods showed good agreement within 1.5%. The proposed image analysis method yielded length estimates with CV from 0.23 to 0.88%, which was lower than the CVs of the line-intersect method. Moreover, the lengths of overlapping samples were calculated correctly because the image analysis method distinguished an overlapping pixel from a thinned image, while the calculation with the line-intersect method showed underestimation because overlaps were not considered in that method. This revised version was published online in June 2006 with corrections to the Cover Date.     

A rapid quantitative measurement of root length and root branching by microcomputer image analysis

A computer program was made for fast and reliable measurement of root length and for estimating the number of root tips and branching points. Image-processing procedures available in a program package for image analysis by means of a personal computer were used. The method is described in this paper and some results of tests on variance and systematic errors (bias) are discussed.Time required for analysis of an evenly spread root (sub-)sample with a total length of max. 300 cm was reduced to less than 20 seconds. Random deviations from the real length, determined by measuring known lengths of wire, did not exceed 5%, after correction for length density dependent bias. Counts of root tips appeared to be unreliable, but branching ratios could be determined fairly accurately, after correction for the length density dependent number of pseudo-branches (e.g. crossings). Rhizotron root photographs were also analysed satisfactorily, after modification of a few steps in the program.     

泡囊丛枝(VA)菌根对玉米根际磷酸酶活性的影响

以玉米为材料,利用三室隔网培养方法,研究了缺P土壤上施用植酸和卵磷脂时接种几种菌根真菌(Glomus mosseae, Glmous versiformea, Gigaspora margarita)对根际土壤酸性磷酸酶和碱性磷酸酶活性的影响.玉米生长70d后,收获测定距根表不同距离土壤中的磷酸酶活性.结果表明,接种菌根真菌增加了根际土壤酸性和碱性磷酸酶活性,Gigaspora margarita菌根菌的作用大于其它2个菌根菌.不同P源对磷酸酶活性有明显影响.     

Prostatic-like acid phosphatase in human endometrial glands and its cyclic activity

Estrogen-induced autocrine and paracrine growth factors are thought to stimulate endometrial proliferation. However, the proliferation is arrested at an early secretory phase although the amount of growth factors and their receptors remains constant. These receptors are protein tyrosine kinases which cause activating receptor autophosphorylation and phosphorylation of signalling substances. One inhibitory mechanism is the reverse dephosphorylation by phosphatases hydrolysing phosphotyrosines. Previously, an acid phosphotyrosine phosphatase activity was found in endometrial secretory glands. The purpose of this study was to evaluate its characteristics. Catalytic and immunohistochemical techniques were applied on sections obtained from human endometrium and other tissues. Endometrial acid phosphatase hydrolysed phosphotyrosine, not only at acid, but also at neutral pH values. An alternative substrate was α-naphthyl phosphate or β-glycerophosphate but not phosphoserine. Activities were inhibited by tartrate and fluoride but not by formaldehyde. These catalytic properties are identical only to those of prostatic acid phosphatase (PAP). A PAP-like nature was also proved by positive PAP immunohistochemistry. In conclusion, endometrial glands contain a phosphotyrosine phosphatase which is identical to PAP. Its activity is menstrual-cycle-dependent, being present only at the secretory phase, and it may counterbalance receptor tyrosine kinases terminating glandular proliferation despite constant levels of growth factors and their receptors.     

Barley genotypes differ in activity of soluble extracellular phosphatase and depletion of organic phosphorus in the rhizosphere soil

In the present investigation we studied the extent of variation among barley genotypes (Hordeum vulgare L. cv. Alexis, Canut, Digger, Etna, Peel) in their ability: i) to induce activity of soluble extracellular phosphatase in rhizosphere soil. ii) to withdraw bicarbonate extractable organic phosphorus (NaHCO₃-P₀). All the genotypes induced 3–4 times higher phosphatase activities in rhizosphere soil as compared to bulk soil. Among the genotypes, there were significant (p>0.01) differences in soluble extracellular and non-soluble phosphatase activities and depletion of NaHCO₃-P₀ in soil near their root mats. Etna induced highest phosphatase activities and depleted most NaHCO₃-P₀ from the rhizosphere soil. A high correlation (r=0.79) was found between the activity of soluble extracellular phosphatase and the quantity of NaHCO₃-P₀ withdrawn from the rhizosphere soil by the barley genotypes.     

泡囊丛枝（VA）菌根对玉米际磷酸酶活性的影响

以玉米为材料,利用三室隔网培养方法,研究了缺P土壤上施用植酸和卵磷脂时接种几种菌根真菌（Glomus mosseae,Glmous versiformea,Gigaspora margarita)对根际土壤酸性磷酸酶和碱性磷酸酶活性的影响,玉米生长70d后,收获测定距根表不同距离土壤中的磷酸酶活性,结果表明,接种菌根真菌增加了根际土壤酸性和碱性磷酸酶活性,Gigaspora margarita菌根菌的作用大于其它2个菌极菌,不同P源对磷酸酶活性有明显影响。     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium

The alkaline phosphatases present on isolated brush border and basal lateral membranes of rat duodenal epitheilum were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6–9.8, similar substrate k_m's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin or papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000–150,000, and both enzymes show an R_f value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The V_max of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 μmoles/mg-h as opposed to 14 μmoles/mg-h. The differences in V_max are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

Automated measurement of root length with a three-dimensional high-resolution scanner and image analysis

For measuring the length of root samples, the use of a three-dimensional (3D) scanner is proposed to address the problem of a too low resolution. The scanner's high resolution (up to 354 pixels per cm) enables in the resulting grey-value image very thin roots (diameter 100 m) to be segmented from the background by a simple thresholding operation. After skeletonizing, total length of the roots is calculated by multiplying the number of skeleton pixels by a correction factor. A comparison with the modified Newman Line-Intersect Method showed a correlation of r=0.98. Besides its superior resolution, an advantage of this type of scanner is its focusing depth, which allows root samples to be recorded on the scanbed similarly to a camera-oriented system.     

Heparin column analysis of serum type 5 tartrate-resistant acid phosphatase isoforms

The objective of the present study was to develop a specific method for the separation of tartrate-resistant acid phosphatase (TRAP) derived exclusively from osteoclasts. Heparin column-bound TRAP in human serum was separated into three peaks of TRAP activity when eluted with a linear gradient of sodium chloride. The last peak corresponded to TRAP 5b which was first named according to its electrophoretic mobility [Clin. Chem. 24 (1978) 309] and was considered to be derived from osteoclasts [J. Bone Miner. Res. 13 (1998) 683]. The second peak was found to be TRAP 5a. The height of the last peak varied from age to age.     

Effect of vanadium compounds on acid phosphatase activity

The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.     

Studies of the purine analog associated modulation of human erythrocyte acid phosphatase activity

Summary The activity of the human erythrocyte acid phosphatase is modulated by a series of structural analogs of purine. The unsubstituted purine base does not affect the enzyme activity. Addition of a substituent at the number six position usually generates an analog which activates the enzyme while similar substitutions at the two position usually generate an inhibitor. Pyrimidines are generally ineffective as modulators while several modifications of the imidazole ring of the purine analogs do not abolish the modulator activity of the purine analog. The level of response to all active analogs is isozyme specific. Differences in apparent relative affinities among the modulators are noted. The modulators with a positive effect on enzyme activity, are effective in the presence of methanol which is more effective than H₂0 as a phosphate acceptor. These analogs act by enhancing the rate of transfer of phosphate to H₂O, while decreasing the rate of transfer to methanol. The results suggest that the purine analogs may act by altering the rate of hydrolysis of the phosphoenzyme intermediate by H₂0 or may change the rate-limiting step in the catalytic mechanism.     

Experimentally induced elevations in acid phosphatase activity in hemolymph of Biomphalaria glabrata (Mollusca).

The levels of acid phosphatase activity in the hemocytes and serum of the following four groups of the pulmonate gastropod Biomphalaria glabrata were ascertained: (1) those injected with heat-killed Bacillus megaterium, (2) those challenged with sterile distilled water, (3) those shaminjected, and (4) those left untampered. It was determined that challenge with heat-killed bacteria sesulted in significant elevations in acid phosphatase activity in both cells and serum at 1, 2, and 4 hr postinjection, with the level being highest at 2 hr. Injection with water resulted in a significant elevation in cellular enzyme level at 1 hr postinjection but not at 2 and 4 hr; however, there were significant elevations at 2 and 4 hr in serum. This is interpreted to indicate that the elevated intracellular enzyme was subsequently released into serum. Sham injection resulted in significant elevations in acid phosphatase levels in hemocytes at 2 and 4 hr postinjection but no increase of enzyme activity in serum during the course of the experiment. This is interpreted to mean that this hydrolase was not released as a result of sham injection. The source of acid phosphatase was apparently the cytoplasmic granules of granulocytes, which are true lysosomes.     

根际微生物调控植物根系构型研究进展

根系构型是最重要的植物形态特征之一,具有可塑性,既由遗传因素控制,又受到许多环境因子的调控。近年的大量研究表明,根际微生物能够调控植物的根系构型,进而影响植物的一系列生理与生态过程。综述丛枝菌根真菌(AMF)、根瘤菌、植物根际促生菌(PGPR)等重要根际微生物类群对植物根系构型的调控模式以及相应的调控机理,并对进一步的研究进行了展望,旨在为今后的相关研究和实际应用提供参考。     

Expression and low pH increase the activity of an apoplastic acid phosphatase in growing tissues from Zea mays

Auxin‐induced secretion of an acid phosphatase (EC 3.1.3.2) leads to the hypothesis that this enzyme may be involved in plant cell elongation growth (W. Pfeiffer. 1996. Physiol. Plant. 98: 773–779). Elongation growth can be characterized by the effects of pH, phosphate and citrate, and the correlation with a particular region of the root: the elongation region. Therefore, it was investigated whether these parameters may reveal further correlations between acid phosphatase and elongation growth. The following results were obtained. (1) An extracellular acid phosphatase with high substrate affinity was characterized (Michaelis‐Menten constant, 0.03 m M for 4‐methylumbelliferyl phosphate; pH optimum, 3.0). The pH dependence of the enzyme was similar to that of elongation growth of coleoptile segments after pretreatment with phosphate (U. Kutschera and P. Schopfer. 1985. Planta 163: 483–493). (2) Phosphate inhibited both the acid phosphatase and coleoptile growth. Phosphate was a competitive inhibitor of the acid phosphatase (inhibitor constant, 2.5 m M ). (3) Citrate inhibited coleoptile growth and the acid phosphatase in a similar way (inhibitor constant, 21 mM). (4) The elongation region of maize roots contained more apoplastic acid phosphatase than adjacent regions (170%). The pH dependence of the enzyme suggests that the low pH reported for the elongation region would result in an additional increase of the enzymatic activity (pH optimum at 3.0).